Use of the KAL protein and treatment with the KAL protein in treatment of retinal, renal, neuronal and neural injury

ABSTRACT

KAL protein is identified the active agent in a therapeutic composition for treatment of injury to nerve tissue, including spinal cord tissue, as well as support of treatment for renal grafts. Additionally, therapeutic treatment of renal injury, and kidney transplantation and renal surgery, is effected by administration of KAL protein. The therapeutic agent may be administered locally, or intravenously. Retinal disorders may be similarly treated.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention pertains to the use of the KAL protein and to thetreatment of patients suffering from neural, retinal and renal insult.

2. Background of the Invention

Kallmann's syndrome (KS) refers to the association of hypogonadism withanosmia (or hyposmia). Hypogonadism in KS is due togonadotropin-releasing hormone (GnRH) deficiency (Naftolin et al., 1971;Sherins and Howards, 1986). Anosmia has been related to the absence orhypoplasia of the olfactory bulbs and olfactory tracts (De Morsier,1954). In animals, the existence of interactions between olfactory andreproductive functions has long been reported (Whitten, 1956 Bruce,1959; McClintock, 1971). More recently, developmental links between theolfactory system and the GnRH neuroendocrine system have also beenidentified. Embryo logical studies in several species including mouse(Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989), monkey(Ronnekleiv and Resko, 1990), chicken (Murakami et al., 1991; Norgrenand Lehman, 1991 Nurakami and Akai, 1996), newt (Murakami et al., 1992)and man (Schwanzel-Fukuda et al., 1995), have led to the conclusion thatGnRH synthesizing neurons migrate from the olfactory epithelium to thebrain during embryonic life. GnRH cells migrate along an olfactoryepithelium-forebrain axis of nerve fibers. In mammals, migrating GnRHcells are primarily found in close association with the vomeronasal andterminal nerves (Schwanzel-Fukuda et al, 1992), whereas in the chickenthey appear to ascend along the olfactory nerves themselves (Murakami etal., 1991). Ultimately, the GnRH neurons reach the preoptic andhypothalamic areas where the neurosecretion takes place. From theseobservations, it was first hypothesized that the “double clinicaldefect” observed in KS affected patients (i.e. hypogonadism and anosmia)could be related to a unique defect in the development process of botholfactory and GnRH neurons.

The study of a human 19 week old male fetus carrying a large Xpdeletion, including the KAL gene responsible for the X-linked form ofthe disease, has shown that neither the GnRH neurons, nor the axonterminals of the olfactory, terminalis and vomeronasal neurons werepresent in the brain. Although GnRH cells and olfactory axons had leftthe olfactory epithelium, they had accumulated in the upper nasal area,on the peripheral side of the dura layer (Schwanzel-Fukuda et al.,1989). This observation indicated that the embryonic defect responsiblefor the X-linked KS did not involve the initial differentiation step ofolfactory and GnRH neurons within the olfactory placode, but rather thesubsequent migration pathway of olfactory axons and GnRH cells to thebrain. Furthermore, some patients have unilateral renal aplasia (Wegenkeet al., 1975).

The human KAL gene has been isolated by positional cloning strategies(Franco et al., 1991; Legouis et al., 1991; Hardelin et al., 1992). Thegene encodes a 680 amino acid putative protein (SwissProt P23352)including a signal peptide. The deduced amino acid sequence provides noevidence for either a hydrophobic transmembrane domain or glycosylphosphatidyl inositol anchorage, suggesting that the protein isextracellular.

The interspecies conservation of the KAL gene sequence has been exploredby Southern blot analysis with human KAL cDNA probes. Crosshybridization was observed in various mammals and in the chicken(Legouis et al., 1993). The KAL orthologue has been isolated in thechicken (Legouis et al., 1993; Rugarli et al., 1993). Sequencecomparison with the human KAL cDNA demonstrated an overall identity of72%, with 75% identity at the protein level.

The expression of the KAL gene during embryonic development has beenstudied in the chicken by in situ hybridization (Legouis et al., 1993;Legouis et al., 1994; Rugarli et al., 1993). From embryonic day 2 (ED2)to ED8, the KAL gene is expressed in various endodermal, mesodermal andectodermal derivatives, whereas from ED8 onwards, the expression isalmost entirely restricted to definite neuronal populations in thecentral nervous system including mitral cells in the olfactory bulbs,Purkinje cells in the cerebellum, striatal, retinal and tectal neurons,most of which still express the gene after hatching. According to such aspatio-temporal pattern of expression, it is proposed that the KAL geneis involved both in morphogenetic events and in neuronal latedifferentiation and/or survival.

SUMMARY OF THE INVENTION

There is no adequate treatment presently available that leads tospecific growth and guidance of neurons which have been injured or havedegenerated.

Surprisingly, the inventors have discovered that the purified KALprotein possess different in vitro biological activities includingneuron growth activity, and neurite fasciculation activity as well asadhesion properties to cerebellar neurons the latter being mediated, atleast in part, via the fibronectin type III of the KAL protein.

In addition the KAL protein is an appropriate substrate for neuronalsurvival. Given these properties, the KAL protein, its receptor(s) andits ligands are relevant to neuronal regeneration:

survival

adhesion

growth

fasciculation

Consequently, an object of the present invention concerns thetherapeutic use of KAL protein or one of its biologically activederivatives, alone or in combination with other ligands, in disease ofcentral or peripheral nervous system including:

One subject of the present invention is a therapeutic compositioncomprising a pharmaceutically active amount of a protein selected amongthe group consisting of:

the purified KAL protein;

a protein having at least 80% homology in aminoacid sequence with theKAL protein; or with protein having at least 80% homology in aminoacidsequence with a purified biologically active part of the KAL protein;

a protein which is specifically recognized by antibodies directedagainst the purified KAL protein.

By <<biologically>> active part of the KAL protein is intended a peptidehaving an aminoacid sequence which is contained in the entire aminoacidsequence of the KAL protein and which peptide exhibits at least one ofthe following in vitro activities

survival activity for cells, and specifically for neurons;

Growth promoting activity for neurons;

induction of neurite fasciculation;

Adhesion function.

A particular biologically active part of the KAL protein consists in oneor several of the four fibronectin type III repeat of the KAL protein(FIG. 9) alone or in combination one with each other that are obtainedby transfection of a procaryotic or an eukaryotic cell, specifically aCHO cell with the corresponding encoding DNA that has been inserted in asuitable expression vector.

Thus, this therapeutic composition according to the present inventioncomprises either the KAL protein or one of its <<biologically activederivatives>> that are above defined.

Another subject of the present invention is a therapeutic compositioncontaining a pharmaceutically effective amount of a polynucleotidesequence (RNA, genomic DNA or cDNA) coding for the purified KAL proteinor a biologically active derivative of the KAL protein.

Another subject of the present invention is a method for cultivatingneuronal cells in vitro comprising the addition of a biologically activeamount of either the purified KAL protein, a protein having at least 80%homology in aminoacid sequence with the KAL protein or a purifiedbiologically active part of the KAL protein to the cell culture medium.

Another subject of the present invention is a method for the productionof the purified recombinant KAL protein comprising the steps of:

a) Cultivating a prokaryotic or an eukaryotic cell that has beentransfected with a vector carrying a DNA insert coding for the KALprotein, a purified biologically active part of the KAL protein or aprotein which is recognized by antibodies directed against the purifiedKAL protein a purified biologically active part of the KAL protein or aprotein which is recognized by antibodies directed against the purifiedKAL protein

b) isolating the recombinant KAL protein from the culture preparation ofthe transfected prokaryotic and eukaryotic cell.

Another subject of the present invention is a method for screeningligands that binds to the KAL protein.

Another subject of the present invention is a method for screeningmolecules that modulates the expression of the KAL protein.

1. Nerve injury of traumatic, infectious, metabolic or inherited origin.

2. Spinal injury of traumatic, infectious, metabolic or inheritedorigin.

3. Retinal disorder graft in context of traumatic, infectious, metabolicor inherited origin.

Renal treatment based on the role of the KAL protein in kidneymorphogenesis:

4. Renal disease, hypoplasia or agenesis of traumatic, infectious,metabolic or inherited origin.

5. Kidney transplantation and renal surgery.

The diseases giving rise to these conditions are varied and include,among others, amyotrophic lateral sclerosis, multiple sclerosis,Parkinson's, injuries of traumatic origin, neurotrophic ulcers, maculardegeneration, diabetes, leprosy and renal failure.

The clinical use of the KAL protein can be administered in the form of asolution, gel or dry powder. It can be introduced locally. It can beadministered intraveneously using devices that overcome the blood brainbarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The KAL protein promotes the adhesion of cerebellar granulecells. Cerebellar granule cells were isolated from postnatal day-5 micewere plated on plastic surfaces which were coated with KAL protein, orwith BSA, or with laminin for 90 min at 37° C. as described in Materialsand Methods. The wells were washed three times with PBS and the adherentcells were counted as described in Materials and Methods. Similarresults were obtained in three separate experiments.

The results are expressed as the percentage of adherent cells, relativeto the total number of cells deposited in the well.

FIG. 2. The KAL protein promotes the adhesion of PC12 cells. PC12 cellswere plated on plastic surfaces which were coated with KAL protein, orwith BSA, or with laminin for 90 min at 37° C. as described in materialsand methods. The wells were washed three times with PBS and adherentcells were counted as described in Materials and Methods.

The results are expressed as the percentage of adherent cells, inrelation to the total number of cells deposited on the substratum.

FIG. 3. Antibody-mediated inhibition of PC12 cell adhesion to the KALprotein. PC12 cells were plated on wells which had been previouslycoated with KAL protein and incubated in the presence of increasingconcentrations of antiserum directed against the KAL protein. The numberof adherent cells was calculated as described above. The results ofthree independent experiments are expressed as the percentage ofadherent cells in presence of immune or preimmune sera, relative to thetotal number of cells deposited in the wells.

FIG. 4. Adhesion of PC12 cells to the KAL protein was inhibited in thepresence of heparin. PC12 cells were added to the wells which had beenpreviously coated with KAL protein and incubated in the presence ofincreasing concentrations of heparin, and then the number of adherentcells was calculated as described above. The results are expressed asthe percentage of adherent cells in absence or in presence of heparin,in relation to the total number of cells deposited on the substratum.

FIG. 5. Adhesion of PC 12 cells to KAL protein was inhibited in thepresence of R1-FNIII PC12 cells were incubated with increasingconcentrations of R1-FNIII, or human serum albumin (HSA) and added tothe wells which have previously been coated with KAL protein. The numberof adherent cells was calculated as described above. The results areexpressed as the percentage of adherent cells in absence or in presenceof R1-FNIII, relative to the total number of cells deposited on thesubstratum.

FIG. 6: Reaggregates of cerebellar neurons from postnatal day-5 arecultured for 48h on KAL protein substrate (A), or respectively onpositive or negative controls, poly-1-lysine (B), BSA (C). Cells werestained with toluidine blue. Note that KAL protein is a permissivesubstrate for survival and neurite outgrowth of cerebellar granulecells.

FIG. 7. Immunodetection of the KAL protein expressed in CHO cells. Wildtype CHO cells (A), KAL-transfected clone 1-1 (B), and 2-3 (C). Cellswere fixed using paraformaldehyde. Note that the immunostainingdelineates the cells and displays the expected pattern for anextracellular matrix component.

FIG. 8. Induction of neurite fasciculation from cerebellar cellaggregates by a monolayer of KAL-expressing cells. Aggregates ofcerebellar neurons from post-natal day 5 mice were cultured for 24 h onmonolayers of either wild type CHO cells (A), or clones ofKAL-transfected CHO cells, clone 2-3 (B-D) and clone 1-1 (E and F).Neurites were short and fasciculated on KAL-expressing cells (B, D andF). C and E: in the presence of anti-KAL Fab fragments (0.2 mg/ml)neurite fasciculation was not induced from cell aggregates cultured onKAL-transfected cells. Neurons were stained for GAP 43 immunoreactivity.

FIG. 9: Aggregates of cerebellar neurons cultured for 24 h on eitherwild type CHO cells (A) or clones of KAL-transfected CHO cells: clone2-3 (B and C). Neurite fasciculation observed on KAL-expressing cells(B) is prevented by the addition of anti-KAL Fab to culture medium (C).

FIGS. 10A, 10B, and 10C: Amino acid sequence of the human KAL protein(SEQ ID NO: 1), the fibronectin type III repeats are respectivelylocated in the following sequences:

the sequence beginning at aminoacid in position 182 and ending ataminoacid in position 286 of the entire aminoacid sequence of the humanKAL protein;

the sequence beginning at aminoacid in position 287 and ending ataminoacid in position 403 of the entire aminoacid sequence of the humanKAL protein;

the sequence beginning at aminoacid in position 404 and ending ataminoacid in position 541 of the entire aminoacid sequence of the humanKAL protein;

the sequence beginning at aminoacid in position 542 and ending ataminoacid in position 662 of the entire aminoacid sequence of the humanKAL protein;

FIG. 11: Schematic representation of the localization of the differentdomains of the KAL protein.

DETAILED DESCRIPTION OF THE INVENTION

The KAL protein has been produced in transfected eukaryotic cells, andspecifically CHO cells. This protein with an approximate molecular massof 100 kDa is N-glycosylated, secreted in the cell culture medium, andwas found to be localized mainly at the cell surface. Therefore, theprotein encoded by the KAL gene is likely to be an extracellular matrixcomponent in vivo.

For the Purpose of the Present Invention:

A <<gene>> refers to the entire DNA portion involved in the synthesis ofa protein. A gene embodies the structural or coding portion which beginsat the 5′ end from the translation start codon (usually ATG) and extendsto the stop (TAG, TGA, or TAA) codon at the 3′ end. It also contains apromoter region, usually located 5′ or upstream to the structural gene,which initiates and regulates the expression of a structural gene. Alsoincluded in a gene are the 3′ end and poly(A)+addition sequences.

A <<structural gene>> is that portion of a gene comprising a DNA segmentencoding a protein, polypeptide or a portion thereof, and excluding the5′ and 3′ non coding sequences. Moreover, since heparin treatment ofcell membrane fractions resulted in the release of the protein, wesuggest that heparan-sulfate proteoglycans are involved in the bindingof the protein to the cell membranes. Polyclonal and monoclonalantibodies directed against the purified protein were generated. Theysubsequently allowed us to determine the cellular distribution of theprotein in the chicken central nervous system at late stages ofembryonic development. The protein is present on cell bodies and alongneurites of definite neuronal populations including Purkinje cells inthe cerebellum, mitral cells in the olfactory bulbs and several neuronalcell populations in the optic tectum and the striatum[Soussi-Yanicostas, 1996].

The N-terminal sequence of the KAL protein is cysteine-rich and can besubdivided into two subregions. The first has no similarity with anyknown protein. The other fits the consensus whey acidic protein (WAP)4-disulfide core motif (Dandekar et al., 1982; Hennighausen and Sippel,1982), a motif shared by several small proteins with serine proteaseinhibitory activity (Kato and Tominaga, 1979; Seemuller et al., 1986;Stetler et al., 1986; Wiedow et al., 1990). A particular feature of theC-terminus of the protein is the presence of 11 basic (including 6histidyl) residues among 20 mostly hydrophilic amino acids. The KALprotein contains four contiguous fibronectin type III repeats (delCastillo et al., 1992). This motif has been found in a wide variety ofmolecules with morphoregulatory roles, most of which are involved incell adhesion, fasciculation and growth of neurons. Among them, L1/NgCAM(Moos et al., 1988; Burgoon et al., 1991) Nr-CAM/Bravo (Grumet et al.,1991; Kayyem et al., 1992), F3/F11 (Gennarini et al., 1989; Brummendorfand Rathjen, 1993), TAG/Axonin-1 (Furley et al., 1990; Zuellig et al,1992), Tenascin-R (Norenberg et al, 1995), Tenascin-C (Gotz et al.,1996). Interestingly, the type III repeats of the protein encoded by theKAL gene show even greater similarity with those of cell adhesionmolecules such as TAG-1/Axonin-1, L1, and F3/F11 (Brummendorf andRathjen, 1993) which have been shown to mediate neurite outgrowth oraxon—axon interactions [Sonderegger and Rathjen, 1992 #48]. Altogether,the sequence comparisons suggest that the protein encoded by the KALgene has several functions including protease inhibitory activity andadhesion.

We demonstrate that the purified KAL protein is a cell adhesion moleculethat is permissive for neuron growth in vitro and is thus particularlysuitable for neuron growth assays in vitro. We also show thattransfected CHO cells producing the KAL protein induce axonalfasciculation of cerebellar granule cells cultivated upon this CHO cellmonolayer.

These results have allowed the inventors to design specific therapeuticcompositions for treating various neuronal or renal disorders using thepurified KAL protein or a biologically active derivative of the KALprotein as described above or, as an alternative embodiment, using apolynucleotide encoding for the KAL protein or for one of itsbiologically active derivative.

In a preferred embodiment of the therapeutic compositions of the presentinvention, the amount of the biologically active peptide component iscomprised in the range from 0.1 μg/ml to 10 μg/ml in the body fluid. Thedose-range is expressed in reference to the bioavailability of the KALprotein or of one of its biologically active derivatives at the bodysite to be treated.

As already mentioned, a particular biologically active part of the KALprotein consists in one or several of the four fibronectin type IIIrepeat of the KAL protein (FIG. 9) alone or in combination one with eachother that are obtained by transfection of a procaryotic or aneukaryotic cell, specifically a CHO cell, with the correspondingencoding DNA that has been inserted in a suitable expression vector.

A suitable vector for the expression of the biologically active part ofthe KAL protein above-defined in baculovirus vector that can bepropagated in insect cells and in insect cell lines. A specific suitablehost vector system is the pVL 1392/1393 baculovirus transfer vector(Pharmingen) that is used to transfect the SF9 cell line (ATCC No. CRL1711) which is derived from Spodoptera frugiperda.

Another suitable vector for the expression in bacteria and in particularin E. coli, is the pQE-30 vector (QIAexpress) that allows the productionof a recombinant protein containing a 6×His affinity tag. The 6×His tagis placed at the C-terminus of the recombinant KAL protein biologicallyactive part which allows a subsequent efficient purification of therecombinant protein by passage onto a Nickel or Copper affinitychromatography column. The Nickel chromatography column may contain theNi-NTA resin (Porath et al., 1975).

In another embodiment of the therapeutic composition according to theinvention, the said composition comprises a polynucleotide coding forthe KAL protein or one of its biologically active derivatives in orderto perform a gene therapy.

The gene therapy consists in correcting a defect or an anomaly(mutation, aberrant expression etc.) by the introduction of a geneticinformation in the affected organism. This genetic information may beintroduced in vitro in a cell that has been previously extracted fromthe organism, the modified cell being subsequently reintroduced in thesaid organism, directly in vivo into the appropriate tissue.

The method for delivering the corresponding protein or peptide to theinterior of a cell of a vertebrate in vivo comprises the step ofintroducing a preparation comprising a pharmaceutically acceptableinjectable carrier and a naked polynucleotide operatively coding for thepolypeptide is taken up into the interior of the cell and has apharmaceutical effect at the renal, retinal or the neuronal level of thevertebrate.

In a specific embodiment, the invention provides a pharmaceuticalproduct, comprising a naked polynucleotide operatively coding for theKAL protein or one of its biologically active derivatives, in solutionin a physiologically acceptable injectable carrier and suitable forintroduction interstitially into a tissue to cause cells of the tissueto express the said protein or polypeptide.

Advantageously, the therapeutic composition containing a nakedpolynucleotide is administered locally, near the site to be treated.

The polynucleotide operatively coding for the KAL protein or one of itsbiologically active derivatives is a vector comprising the genomic DNAor the complementary DNA coding for the KAL protein or its proteinderivative and a promoter sequence allowing the expression of thegenomic DNA or the complementary DNA in the desired vertebrate cells.

The vector component of a therapeutic composition according to thepresent invention is advantageously a plasmid, a part of which is ofbacterial origin, which carries a bacterial origin of replication and agene allowing its selection such as an antibiotic resistance gene.

By <<vector>> according to this specific embodiment of the invention isintended a circular or linear DNA molecule.

This vector may also contain an origin of replication that allows it toreplicate in the vertebrate host cell such as an origin of replicationfrom a bovine papillomavirus.

The promoter carried by the said vector is advantageously thecytomegalovirus promoter (CMV). Nevertheless, the promoter may also beany other promoter with the proviso that the said promoter allow anefficient expression of the DNA insert coding for the KAL protein or oneof its biologically active derivatives within the host.

Thus, the promoter is selected among the group comprising:

an internal or an endogenous promoter, such as the natural promoterassociated with the structural gene coding for KAL; such a promoter maybe completed by a regulatory element derived from the vertebrate host,in particular an activator element;

a promoter derived from a cytoskeletal protein gene such as the desminpromoter (Bolmont et al., J. of Submicroscopic cytology and pathology,1990, 22:117-122; Zhenlin et al., Gene, 1989, 78:243-254).

As a general feature, the promoter may be heterologous to the vertebratehost, but it is advantageously homologous to the vertebrate host.

By a promoter heterologous to the vertebrate host is intended a promoterthat is not found naturally in the vertebrate host.

Therapeutic compositions comprising a naked polynucleotide are describedin the PCT application No. WO 90/11092 (Viacl Inc.) and also in the PCTapplication No. WO 95/11307 (Institut Pasteur, INSERM, Universitéd'Ottawa) as well as in the articles of Tacson et al. (1996, NatureMedicine, 2(8):888-892) and of Huygen et al. (1996, Nature Medicine,2(8):893-898).

The therapeutic compositions described above may be administered to thevertebrate host by a local route such as an intramuscular route.

The therapeutic naked polynucleotide according to the present inventionmay be injected to the host after it has been coupled with compoundsthat promote the penetration of the therapeutic polynucleotide withinthe cell or its transport to the cell nucleus. The resulting conjugatesmay be encapsulated in polymer microparticles as it is described in thePCT application No. WO 94/27238 (Medisorb Technologies International).

In another embodiment, the DNA to be introduced is complexed withDEAE-dextran (Pagano et al., 1967, J. Virol., 1:891) or with nuclearproteins (Kaneda et al., 1989, Science 24:375), with lipids (Feigner etal., 1987, Proc. natl. Acad. Sci., 84:7413) or encapsulated withinliposomes (Fraley et al., 1980, J. Biol. Chem., 255:10431).

In another embodiment, the therapeutic polynucleotide may be included ina transfection system comprising polypeptides that promote itspenetration within the host cells as it is described in the PCTapplication WO 95/10534 (Seikagaku Corporation).

The therapeutic polynucleotide and vector according to the presentinvention may advantageously be administered in the form of a gel thatfacilitates their transfection into the cells. Such a gel compositionmay be a complex of poly-L-Lysine and lactose, as described by Midoux(1993, Nucleic Acids Research, 21:871-878) or also poloxamer 407 asdescribed by Pastore (1994, Circulation, 90:I-517). The therapeuticpolynucleotide and vector according to the invention may also besuspended in a buffer solution or be associated with liposomes.

Thus, the therapeutic polynucleotide and vector according to theinvention are used to make pharmaceutical compositions for deliveringthe DNA (genomic DNA or cDNA) coding for the KAL protein or one of itsbiologically active derivatives at the site of the injection.

The amount of the vector to be injected varies according to the site ofinjection and also to the kind of disorder to be treated. As anindicative dose, between 0.1 and 100 μg of the vector will be injectedin a patient.

In another embodiment of the therapeutic polynucleotide according to theinvention, this polynucleotide may be introduced in vitro in a hostcell, preferably in a host cell previously harvested from the patient tobe treated and more preferably a somatic cell such as a muscle cell, arenal cell or a neurone, in a subsequent step, the cell that has beentransformed with the therapeutic nucleotide coding for the KAL proteinor one of its biologically active derivative is implanted back into thepatient body in order to deliver the recombinant protein within the bodyeither locally or systematically.

In a preferred embodiment, gene targeting techniques are used tointroduce the therapeutic polynucleotide into the host cell. One of thepreferred targeting techniques according to the present inventionconsists in a process for specific replacement, in particular bytargeting the KAL protein encoding DNA, called insertion DNA, comprisingall or part of the DNA structurally encoding for the KAL protein or oneof its biologically active derivatives, when it is recombined with acomplementing DNA in order to supply a complete recombinant gene in thegenome of the host cell of the patient, characterized in that:

the site of insertion is located in a selected gene, called therecipient gene, containing the complementing DNA encoding the KALprotein or one of its biologically active derivatives and in that

the polynucleotide coding for the KAL protein or one of its biologicallyactive derivatives may comprise:

<<flanking sequences>> on either side of the DNA to be inserted,respectively homologous to two genomic sequences which are adjacent tothe desired insertion site in the recipient gene.

the insertion DNA being heterologous with respect to the recipient gene,and

the flanking sequences being selected from those which constitute theabovementioned complementing DNA and which allow,k as a result ofhomologous recombination with corresponding sequences in the recipientgene, the reconstitution of a complete recombinant gene in the genome ofthe eukaryotic cell.

Such a DNA targeting technique is described in the PCT patentapplication No. WO 90/11354 (Institut Pasteur).

Such a DNA targeting process makes it possible to insert the therapeuticnucleotide according to the invention behind an endogenous promoterwhich has the desired functions (for example, specificity of expressionin the selected target tissue).

According to this embodiment of the invention, the inserted therapeuticpolynucleotide may contain, between the flanking sequences and upstreamfrom the open reading frame encoding the KAL protein (or one of itsbiologically active derivatives), a sequence carrying a promotersequence either homologous or heterologous with respect to the KALencoding DNA. The insertion DNA may contain in addition, downstream fromthe open reading frame and still between the flanking sequences, a genecoding for a selection agent, associated with a promoter making possibleits expression in the target cell.

According to this embodiment of the present invention, the vector usedalso contains a bacterial origin of replication of the type colE1,pBR322, which makes the clonings and preparation in E. coli possible. Apreferred vector is the plasmid pGN described in the POT application No.WO 90/11354.

Other gene therapy methods than those using homologous recombination mayalso be used in order to allow the expression of a polynucleotideencoding the KAL protein or one of its biologically active derivativeswithin a patient's body.

In all the gene therapy methods that may be used according to thepresent invention, different types of vectors are utilized.

In one specific embodiment, the vector is derived from an adenovirus.Adenoviruses vectors that are suitable according to the gene therapymethods of the present invention are those described by Feldman and Steg(1996, Medicine/Sciences, synthese, 12:47-55) or Ohno et al. (1994,Sciences, 265:781-784) or also in the french patent application No.FR-94.03.151 (Institut Pasteur, Inserm). Another preferred recombinantadenovirus according to this specific embodiment of the presentinvention is the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or anadenovirus of animal origin (French patent application No. FR-93.05954).

Among the adenoviruses of animal origin it can be cited the adenovirusesof canine (CA V2, strain Manhattan or A26/6 [ATCC VR-800]), bovine,murine (Mavl, Beard et al., 1980, Virology, 75:81) or simian (SAV).

Preferably, the inventors are using recombinant defective adenovirusesthat may be prepared following a technique well-known by one of skill inthe art, for example as described by Levrero et al., 1991, Gene,101:195) or by Graham (1984, EMBO J., 3:2917) or in the European patentapplication No. EP-185.573. Another defective recombinant adenovirusthat may be used according to the present invention, as well as apharmaceutical composition containing such a defective recombinantadenovirus, is described in the PCT application No. WO 95/14785.

In another specific embodiment, the vector is a recombinant retroviralvector, such as the vector described in the PCT application No. WO92/15676 or the vector described in the PCT application No. WO 94/24298(Institut Pasteur). The latter recombinant retroviral vector comprises:

a DNA sequence from a provirus that has been modified such that:

the gag, pol and env genes of the provirus DNA has been deleted at leastin part in order to obtain a proviral DNA which is incapable ofreplicate, this DNA not being able to recombine to form a wild virus;

the LTR sequence comprises a deletion in the U3 sequence, such that themRNA transcription that the LTR controls is significantly reduced, forexample at least 10 times, and

the retroviral vector comprises in addition an exogenous nucleotidesequence coding for the KAL protein or one of its biologically activederivatives under the control of an exogenous promoter, for example aconstitutive or an inductible promoter.

By exogenous promoter in the recombinant retroviral vector describedabove is intended a promoter that is exogenous with respect to theretroviral DNA but that may be endogenous or homologous with respect tothe KAL protein entire or partial nucleotide coding sequence.

In the case in which the promoter is heterologous with respect to theKAL protein entire or partial nucleotide coding sequence, the promoteris preferably the mouse inductible promoter Mx or a promoter comprisinga tetracyclin operator or also a hormone regulated promoter. A preferredconstitutive promoter that is used is one of the internal promoters thatare active in the resting fibroblasts such the promoter of thephosphoglycerate kinase gene (PGK-1). The PGK-1 promoter is either themouse promoter or the human promoter such as described by Adra et al.(1987, Gene, 60:65-74). Other constitutive promoters may also be usedsuch that the beta-actin promoter (Kort et al., 1983, Nucleic AcidsResearch, 11:8287-8301) or the vimentin promoter (Rettlez and Basenga,1987, Mol. Cell. Biol., 7:1676-1685).

A preferred retroviral vector used according to this specific embodimentof the present invention is derived from the Mo-MuLV retrovirus (WO94/24298).

In one preferred embodiment, the recombinant retroviral vector carryingthe therapeutic nucleotide sequence coding for the KAL protein or one ofits biologically active derivatives is used to transform mammaliancells, preferably autologous cells from the mammalian host to betreated, and more preferably autologous fibroblasts from the patient tobe treated. The fibroblasts that have been transformed with theretroviral vector according to the invention are reimplanted directly inthe patient's body or are seeded in a preformed implant before theintroduction of the implant colonized with the transformed fibroblastswithin the patient's body. The implant used is advantageously made of abiocompatible carrier allowing the transformed fibroblasts to anchorassociated with a compound allowing the gelification of the cells. Thebiocompatible carrier is either a biological carrier, such as coral orbone powder, or a synthetic carrier, such as synthetic polymer fibres,for example polytetrafluoroethylene fibres.

An implant having the characteristics as defined above is the implantdescribed in the PCT application No. WO 94/24298 (Institut Pasteur).

Another subject of the present invention is a method for screeningligands that bind to the KAL protein.

Such a screening method, in one embodiment, comprises the steps of:

a) Preparing a complex between the KAL protein and a ligand that bindsto the KAL protein by a method selected among the followings:

preparing a tissue extract containing the KAL protein putatively boundto a natural ligand;

bringing into contact the purified KAL protein with a solutioncontaining a molecule to be tested as a ligand binding to the KALprotein.

b) visualizing the complex formed between the KAL protein from thetissue extract and the natural ligand of the KAL protein or the complexformed between the purified KAL protein and the molecule to be tested.

For the purpose of the present invention, a ligand means a molecule,such as a protein, a peptide, a hormone, or antibody or a syntheticcompound capable of binding to the KAL protein or one of itsbiologically active derivatives or to modulate the expression of thepolynucleotide coding for the KAL protein or coding for one of itsbiologically active derivatives.

In the first embodiment of the screening procedure wherein a naturalligand of the KAL protein is to be characterized, it is processed asfollows:

A tissue putatively containing the KAL protein bound to its naturalligand, for example the cerebellum, olfactory bulbs, tectum or liverfrom embryos, especially chicken embryos, are homogenized in 10 mMHepes, pH 7.4, containing 100 μg/ml PMSF, 200 μg/ml aprotinin and 5μg/ml Dnase, with a glass-Teflon homogenizer. The homogenate iscentrifuged at 1,000 g for 10 minutes; the supernatant is removed andcentrifuged at 190,000 g for 30 min at 4° C. The pellet containing themembrane fraction is stored at −20° C. until used.

The cell membrane fractions are incubated first in 0.9% Triton X-100,0.1% ovalbumin, 5 mM EDTA, 50 mM Tris-HCl, pH 8, with the P34 immuneserum (Soussi-Yanicostas et al., 1996) overnight at 4° C., then withProtein G-sepharose (Pharmacia) for 2 hours. Complexes are centrifuged,washed three times in PBS and three times in 50 mM Tris-Hcl, pH 8. Thenthe complexes are dissociated in a dissociating buffer containing SDS inorder to dissociate the KAL protein from its bound natural ligand.Immunoprecipitates are analysed by western blot following the techniquedescribed by Gershoni and Palade (1983, Anal. Biochem., 131:1-15). Theanti-KAL protein monoclonal antibody produced by the hybridome clone 1-4was used to detect the KAL protein and a panel of candidate antibodies,for example antibodies directed against different sub-units of integrinsare used (at a concentration of 1.5 μg/ml) to identify the ligand thatwas previously bound to the KAL protein in the tissue extract. IgGperoxidase-conjugated antibody (Bio-Rad, 1/6,000 dilution) is used assecond antibody. The blots are revealed by chemiluminescence with theECL kit (Amersham France).

In a second embodiment of the ligand screening method according to thepresent invention, a biological sample or a defined molecule to betested as a putative ligand of the KAL protein is brought into contactwith the purified KAL protein, for example the purified recombinant KALproduced by the clone CH KAL 2-3/dl, in order to form a complex betweenthe KAL protein and the putative ligand molecule to be tested. Thebiological sample may be obtained from a cerebellum or a renal extract,for example.

When the ligand source is a biological sample, the complexes areprocessed as described above in order to identify and characterize theunknown ligand.

When the putative ligand is a defined known molecule to be tested, thecomplexes formed between the KAL protein and the molecule to be testedare not dissociated prior to the western blotting in order to allow thedetection of the complexes using polyclonal or monoclonal antibodiesdirected against the KAL protein.

In a particular embodiment of the screening method, the putative ligandis the expression product of a DNA insert contained in a phage vector(Parmley and Smith, Gene, 1988, 73:305-318). According to thisparticular embodiment, the recombinant phages expressing a protein thatbinds to the immobilized KAL protein is retained and the complex formedbetween the KAL protein and the recombinant phage is subsequentlyimmunoprecipitated by a polyclonal or a monoclonal antibody directedagainst the KAL protein.

According to this particular embodiment, a ligand library is constructedin recombinated phages from human of chicken genomic DNA or cDNA. Oncethe ligand library in recombinant phages has been constructed, the phasepopulation is brought into contact with the immobilized KAL protein. Thepreparation of complexes is washed in order to remove thenon-specifically bound recombinant phages. The phages that bindspecifically to the KAL protein are then eluted by a buffer (acid pH) orimmunoprecipitated by the monoclonal antibody produced by the hybridomaanti-KAL, clone 1,4, and this phage population is subsequently amplifiedby an over-infection of bacteria (for example E. coli). The selectionstep may be repeated several times, preferably 2-4 times, in order toselect the more specific recombinant phage clones. The last stepconsists in characterizing the protein produced by the selectedrecombinant phage clones either by expression in infected bacteria andisolation, expressing the phage insert in another host-vector system, orsequencing the insert contained in the selected recombinant phages.

One group of the numerous candidate ligands that may be screened belongto the integrin protein family.

Another subject of the present invention is a method for screeningmolecules that modulate the expression of the KAL protein. Such ascreening method comprises the steps of:

a) cultivating a prokaryotic or an eukaryotic cell that has beentransfected with a nucleotide sequence encoding the KAL protein, placedunder the control of its own promoter;

b) bringing into contact the cultivated cell with a molecule to betested;

c) quantifying the expression of the KAL protein.

Using DNA recombinant techniques well known by the one skilled in theart, the KAL protein encoding DNA sequence is inserted into anexpression vector, downstream from its promoter sequence, the saidpromoter sequence being described by Cohen-Salmon et al. (1995, Gene,164:235-242).

The quantification of the expression of the KAL protein may be realizedeither at the mRNA level or at the protein level. In the latter case,polyclonal or monoclonal antibodies may be used to quantify the amountsof the KAL protein that have been produced, for example in an ELISA or aRLA assay.

In a preferred embodiment, the quantification of the KAL mRNA isrealized by a quantitative PCR amplification of the cDNA obtained by areverse transcription of the total mRNA of the cultivatedKAL-transfected host cell, using a pair of primers specific for KAL ofthe kind that are described in the PCT application No. WO 93/02267(Institut Pasteur, HHS).

As an illustrative example, a pair of primers used to quantitate KALreverse-transcribed mRNA is the following:

Primer 1 (SEQ ID NO: 2): 5′ CAG CCA ATG GTG CGG CCT CCT GTC C3′

Primer 2 (SEQ ID NO: 3): 5′ TCC CGG CAG ACA GCG ACT CCGT 3′

The process for determining the quantity of the cDNA corresponding tothe KAL mRNA present in the cultivated KAL-transfected cells ischaracterized in that:

1) a standard DNA fragment, which differs from the KAL cDNA fragment,obtained by the reverse transcription of the KAL-mRNA, but can beamplified with the same oligonucleotide primers is added to the sampleto be analyzed containing the KAL-cDNA fragment, the standard DNAfragment and the KAL-cDNA fragment differing in sequence and/or size bynot more than approximately 10%, and preferably by not more than 5nucleotides by strand,

2) the KAL-cDNA fragment and the standard DNA fragment are coamplifiedwith the same oligonucleotide primers, preferably to saturation of theamplification of the KAL-cDNA fragment,

3) to the reaction medium obtained in step 2), there are added:

either two types of labeled oligonucleotide probes which are eachspecific for the KAL-cDNA fragment and the standard DNA fragment anddifferent from said oligonucleotide primers of step 2), and one of moreadditional amplification cycle(s) with said labeled oligonucleotideprimer(s) is/are performed, so that, during a cycle, after denaturationof the DNA, said labeled oligonucleotide primer(s) hybridize(s) withsaid fragments at a suitable site in order than a elongation with theDNA polymerase generates labeled DNA fragments of different sizes and/orsequences and/or with different labels according to whether theyoriginate from the DNA fragment of interest or the standard fragment,respectively, and then 4) the initial quantity of KAL-cDNA fragment isdetermined as being the product of the initial quantity of standard DNAfragment and the ratio of the quantity of amplified KAL-cDNA fragment,which ratio is identical to that of the quantities of the labeled DNAfragments originating from the amplified KAL-cDNA fragment,respectively, obtained in step 3.)

Primers and probes hybridizing with teh KAL-cDNA fragment and used inthe above-described quantitative PCR amplifications reaction aredescribed in the PCT application No. WO 93/07267 9institut pasteur,HHS).

More techanical details regarding the performing of the quantitative PCRamplification reaction are found in the PCT application No. WO 93/10257(Institut Pasteur, Inserm).

Materials and Methods

Antibodies

Immunoglobulins from pre-immune and anti-human Kal rabbit sera werepurified by affinity chromatography on protein-A sepharose (PharmaciaBiotech., Sweden). Fragments with an antigen-binding site (Fab) wereprepared by proteolytic digestion with papain-agarose (Sigman, USA),undigested IgG were eliminated by protein-A sepharose chromatography andFab were extensively dialyzed against PBS.

Cell Culture

All the culture media, fetal calf serum (FCS) and horse serum werepurchased from Life Technologies (France).

Recombinant CHO cell lines. The 2,4 kb EcoRI insert from the Blue scriptplasmid p85 (Legouis et al., 1991, Cell, 67:423-435) consisting of theentire 2,040 bp coding region of the human KAL cDNA (GenBank accessionnumber M97252), as well as 56 bp and 293 bp of the 5′ and 3′ non codingregions, respectively, was introduced, downstream of the CMV/T7promoter, into a modified pFR400 vector (Genentech Inc., San Francisco,Calif.), pFRCM, that contains a mouse mutant dihydrofolate reductase(dhfr) cDNA. The above-defined p85B plasmid contains a cDNA having thesequence of FIG. 9 and has been deposited at the CNCM (CollectionNationale de Cultures de microorganismes) on Sep. 26, 1991 under theaccession number No. I-1146. This pFRCM-KAL construct was transfectedinto dhfr+CHO cells by calcium phosphate precipitation (Wigler et al.,1979, Cell, 16:777-785). CHO cells were cultivated in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 8% fetal calf serum9Jacques Boy, France). Several independent clones producing KALc wereobtained by stepwise selection in increasing concentrations ofmethotrexate (from 0.3 μM to 1 mM) as previously described 9Kaufman andSharp, 1982, J. Mol. Biol., 159:301-621). Expression of KALc wasassessed at each step by immunocytolabeling using a polyclonal antibodythat has been prepared against the human KAL protein. CloneCHKAL2-3/d11, which is a subclone of the clone CHO-CAL 2.3 wasspecifically selected. The parental CHO cell line and the humanKal-transfected CHO clones (1-1 and 2-3) were maintained in DMEMsupplemented with 8% FCS, 50 UI/ml penicillin and 50 μg/ml streptomycin.

Cerebellar cell cultures. Dissociated cell cultures were obtained fromSwiss mouse cerebella on postnatal day 5. At this age, granule cellsaccount for up to 90% of the total cell population, glial cellsincluded. Cells were dissociated by combined trituration andtrypsinisation, and grown in chemically defined medium DMEM/Ham's F12 (3vol/1 vol) containing 0.2 mM glutamine, 5 μg/ml insulin, 100 μg/mltranferrin, 20 nM progesterone, 100 mM purrescine, 30 nM selenium 100U/ml penicillin and 0.1 mg/ml streptomycin.

Reaggregate cultures of cerebellar neurons from mice on postnatal day-5were prepared according to Gao et al. (1995). After dissocation, cellswere further purified by preplating on a poly-L-Lysine treated (25μg/ml) substrate for 30 min and plated in uncoated 96-well dishes (5 10⁵cells/well) in BME plus 10% horse serum, 5% fetal calf serum, 9 mg/mlglucose, 0.3 mg/ml glutamine, 50 U/ml penicillin and 0.1 mg/mlstreptomycin. Aggregates (100-200 cells) were harvested after 24 h to beused in coculture experiments.

Parental and transfected CHO cells (clones 1.1 and 2.3) were seeded inNunc 8-well labtek slides at a density of 10⁴ cells/well. Cells weregrown for 24 h until confluency and used as monolayer underlyingaggregated cerebellar neurons. Cocultures were established by addingapproximately 50 aggregates/labtek well, and maintained for 24 h or 48 hin defined medium prior to fixation and immunostaining. Where indicated,pre-immune or anti-KAL Fab fragments at a concentration of 0.2 mg/mlwere included for the entire coculture period.

Indirect immunofluorescence. For the visualization of neurons grown onmonolayers, cells were fixed with 4% paraformaldehyde in phosphatebuffer salline (PBS) for 15 min, permeabilized with methanol/acetone for2 min, rehydrated in PBS, incubated with anti-GAP 43 antiserum (Williamset al., 1992, J. Cell Biol. 119 p. 885-892) diluted (1:500) in PBScontaining 3% bovine serum albumin (BSA) for 1 h, rinsed with PBS,incubated with Texas-red conjugated anti-rabbit immunoglobulin (specificfor Fc fragment) diluted (1:100) in PSB containing 3% BSA for 1 h. Afterwashing with PBS, cells were mounted in Mowiol (Calbiochem, USA).Recombinant KAL protein expressed by clones 1.1 and 2.3 was labeled withanti-KAL IgG (dilution 10 μg/ml) after cell fixation with 4%paraformaldehyde in PBS for 15 min and using the same immunofluorescentstaining procedure.

Production and purification of KAL protein The KAL protein was purifiedfrom CHKAL2-3/d11 cells by a three step procedure including twochromatographies. The cells were washed in Ca²⁺and Mg² -free PBS andincubated for 30 min in DMEM supplemented with 350 mM NaCl. The cellsupernatant was supplemented with 0.5% of3-((3-cholamidopropyl)-dimethylammonio)-1-propane-sulfonate (CHAPS), 50μg/ml phenylmethylfulfonyl fluoride (PMSF), 100 μg/ml pepstatin and 100μg/ml leupeptin, and then loaded onto a heparin-Sepharose column(HiTRAP™ Heparin, Pharmacia). NaCl elution fractions were loaded onto animmobilized copper adsorption chromatography column (HiTRAp™ chelatingCU²⁺, Pharmacia) and the protein was eluted as a single peak at 75 mMimidazole.

Adhesion Assay

24-well microtiter plates were coated at 37° C. overnight with 20 μg/mlof laminin, 5 μg/ml of KAL in PBS, pH=7.4. The plates were washed twicewith PBS and non specific sites were blocked by the addition of 1% BSAin PBS for 1 hour at 37° C. Wells were washed twice with PBS. Cerebellarneurons or PC12 cells were resuspended in DMEM to a final concentrationof 10⁶ cells/ml. 500 μl of this suspension was added to each coatedwell. Cells were also added to control wells that had been coated withBSA alone. Plates were incubated at 37° C. for 90 min in a 5% CO2humidified atmosphere. The wells were washed gently twice with 0.5 mlPBS. To remove adherent cells from the wells, 0.5 ml of 0.05%trypsin-EDTA were added to each well. After 10 min at 37° C., the 0.5 mlof trypsin-EDTA containing the detached cells were removed and thenumber of cells was determined by using a cell counter (Coutler, ZMequipped with a Coultronic 256 channelizer).

Each cell adhesion assay was carried out in triplicate. The ration ofadherent cells with respect to the total number of cell×100 wasdetermined as the % of cell adhesion.

Antibodies Inhibition Assays

For inhibition of cell adhesion, 5×10⁵ PC12 cells were deposited onareas previously coated with KAL and with antiserum directed against thehuman KAL protein at different concentrations and treated as describedfor adhesion assay. Each inhibition assay was performed three times inthree independent experiments.

Heparin Inhibition Assays

PC12 cells (Greene et al., 1076, Proc. Natl. Acad. Sci. USA, 73:2424-2428) were added to the wells coated with the KAL protein in thepresence of different concentrations of heparin and treated as describedfor adhesion assays. The assays were performed in triplicate.

Competitive Inhibition of KAL-Mediated Adhesion with Fusion Protein

Human serum albumin fusion protein covering the first repeat offibronectin type III of KAL protein (R1-FNIII) was produced in yeast.The PC12 cells were incubated with different concentrations of R1-FN111,or with Human Serum Albumin (HSA), or with PBS, for 30 min at 37° C. andadded to wells which were coated with KAL protein (5 μg/ml) as describedabove. The assays were performed in triplicate.

Results

It has been hypothesized that the KAL protein mediates cell adhesionbecause of its structural similarity with well characterized celladhesion molecules described by Edelman and Crossin, in 1991. In orderto test this hypothesis, we examined the ability of the KAL proteincoated on a plastic surface to promote adhesion of cerebellar granuleneurons and PC12 cells.

KAL protein isolated from transfected CHO cells was purified by twosuccessive chromatographies on heparin-Sepharose and immobilized copperadsorption columns [Soussi-Yanicostas, 1996 #45] and the purifiedprotein was coated onto Petri dishes. Laminin and bovine serum albumin(BSA) were used as positive and negative controls, respectively.Dissociated mouse cerebellar cells were plated on dishes coated witheither KAL protein or laminin, or BSA. After a 90 minute incubation, 80%of the cerebellar neurons were found to adhere to the KAL coatedsurface. A similar percentage of cell adhesion was observed withlaminin-coated dishes. In contrast, no adhesion was detected on BSASubstrate (FIG. 1). Similar results were observed using PC12 cells (FIG.2). A maximum percentage of cell adhesion was obtained with aconcentration of 5 μg/ml of KAL protein (results not shown).

These data suggest that both cerebellar neurons and PC12 cells have theability to adhere to KAL substrate.

In order to verify that the KAL protein plays a specific role in thiscell adhesion, an adhesion assay was performed in the presence of anantiserum directed against the human KAL protein in the culture medium.As shown in FIG. 3, the addition of anti-KAL antibodies inhibits theadhesion of the PC12 cells to KAL-coated dishes. In contrast, theaddition of pre-immune serum to the adhesion assay, had no effect on theadhesion of PC12 cells to the KAL protein (FIG. 3).

To test whether the interactions of neural cells with KAL protein can beinhibited by addition of soluble glycosaminoglycans, we tested theability of PC12 cells to adhere to KAL substrates in the presence ofheparin. We observed that adhesion of PC12 cells to KAL protein wasinhibited from 0.03 mg/ml of heparin (FIG. 4). These results suggestthat heparan-sulfateproteoglycans may be involved in the PC12 celladhesion to KAL protein.

To investigate the involvement of different domains of KAL protein inPC12 cell adhesion, we produced a human serum albumin fusion proteincontaining the first repeat of fibronectin type III of the KAL protein(R1-FNIII) in yeast, corresponding, from N-terminal end to C-terminalend, to the aminoacid sequence beginning at the aminoacid at position182 from the sequence of FIG. 9 and ending at the aminoacid at position286 from the sequence of FIG. 9. Increasing concentrations of R1-FNIIIwere incubated with PC12 cells for 30 min at 37° C. before adhesionassays on KAL protein. We observed that R1-FNIII perturbs partially theadhesion of PC12 cells to KAL protein (FIG. 5).

In summary, the cell adhesion assays demonstrated that the KAL proteincontains binding sites for molecules present at the cell surface of bothcerebellar neurons and PC12 cells. The adhesion of neural cells to KALprotein may depend on glycosaminoglycans. The fiirst fibronectin typeIII domain of the KAL protein partially account for the binding activityof the molecule.

The Purified KAL Protein is a Permissive Substrate for Neurite Outgrowthof Cerebellar Neurons.

In order to determine the role of purified KAL protein on neuriteoutgrowth, we used granule cell aggregates as a model, prepared asdescribed in the Materials and Methods section. Cerebellar granuleneurons were seeded on surfaces that had been coated with KAL protein.Polylysine and bovine serum albumin (BSA) were used as positive andnegative controls respectively. When aggregates were cultured for 48hours on KAL protein, neurons remained tightly aggregated and displayeda large halo of neuritic processes (FIG. 6A). A similar observation wasobtained on the polylysine-coated surface (FIG. 6B). In contrast, noneuronal survival was observed on the BSA-coated surface (FIG. 6C).

These results show that the KAL protein is a permissive substrate forsurvival and neurite outgrowth of cerebellar granule neurons.

KAL Immunofluorescencestaining at the Surface of Transfected CHO Cells

The different human KAL-expressing CHO cell lines were labeled byindirect immunofluorescence using an antiserum directed against thehuman KAL gene product. Large amounts of the KAL protein were observedat the cell surface of clonal KAL transfected cell lines 1-1 and 2-3(FIG. 7).

Induction of Neurite Fasciculation from Granule Cell Aggregates byKAL-Expressing Cells

Granule cell aggregates from post-natal day-5 mice were grown in definedmedium onto monolayers of CHO cells. After 24 h of coculture, aggregateshad produced long, sinuous, and unfasciculated processes onto controlcells (FIGS. 8A and 9A). By contrast, aggregates grown ontoKAL-expressing cells displayed short, radial and highly fasciculatedneurites (FIGS. 8B and 9B). To ensure that this effect was not anartifact of one particular KAL-expressing cell line, two independentclones (1-1 and 2-3) were tested. They were producing equivalent amountsof the transfected protein as assayed by Western blot. These two clonesexhibited the same ability to both fasciculate and reduce length of theneuritic processes growing from granule cell aggregates (FIGS. 8D andF).

Antibody Reversal of KAL-Induced Neurite Fasciculation from Granule CellAggregates

In order to demonstrate the specificity of Kal's effect on fasciculationand growth inhibition of neurites, anti-KAL fragments (0.2 mg/ml) wereincluded during the entire time of coculture of KAL-expressing cells andgranule cell aggregates. KAL-expressing monolayers displayed intensestaining with anti-KAL Fab as revealed with Texas-red conjugated IgGspecific anti-rabbit antibody (same as FIGS. 7B and C, not shown). Bothantibodies directed against human KAL and the neuronal marker GAP-43have been raised in rabbit. Thus, to avoid monolayer staining, neuronswere visualized using antiGAP-43 and Fc-specific Texas red conjugatedanti-rabbit antibody.

In the presence of anti-KAL Fab bound to the KAL-expressing cellmonolayers, granule cell aggregates showed long and defasciculatedneurites (FIGS. 8C, E). Some long neurites were induced to growcircumferentially instead of radially (FIG. 8C). The presence ofpre-immune Fab had no effect on the fasciculation and growth inhibitionof neurites observed on KAL-expressing CHO cells.

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3 680 amino acids amino acid single linear protein 1 Met Val Pro Gly ValPro Gly Ala Val Leu Thr Leu Cys Leu Trp Leu 1 5 10 15 Ala Ala Ser SerGly Cys Leu Ala Ala Gly Pro Gly Ala Ala Ala Ala 20 25 30 Arg Arg Leu AspGlu Ser Leu Ser Ala Gly Ser Val Gln Arg Ala Pro 35 40 45 Cys Ala Ser ArgCys Leu Ser Leu Gln Ile Thr Arg Ile Ser Ala Phe 50 55 60 Phe Gln His PheGln Asn Asn Gly Ser Leu Val Trp Cys Gln Asn His 65 70 75 80 Lys Gln CysSer Lys Cys Leu Glu Pro Cys Lys Glu Ser Gly Asp Leu 85 90 95 Arg Lys HisGln Cys Gln Ser Phe Cys Glu Pro Leu Phe Pro Lys Lys 100 105 110 Ser TyrGlu Cys Leu Thr Ser Cys Glu Phe Leu Lys Tyr Ile Leu Leu 115 120 125 ValLys Gln Gly Asp Cys Pro Ala Pro Glu Lys Ala Ser Gly Phe Ala 130 135 140Ala Ala Cys Val Glu Ser Cys Glu Val Asp Asn Glu Cys Ser Gly Val 145 150155 160 Lys Lys Cys Cys Ser Asn Gly Cys Gly His Thr Cys Gln Val Pro Lys165 170 175 Thr Leu Tyr Lys Gly Val Pro Leu Lys Pro Arg Lys Glu Leu ArgPhe 180 185 190 Thr Glu Leu Gln Ser Gly Gln Leu Glu Val Lys Trp Ser SerLys Phe 195 200 205 Asn Ile Ser Ile Glu Pro Val Ile Tyr Val Val Gln ArgArg Trp Asn 210 215 220 Tyr Gly Ile His Pro Ser Glu Asp Asp Ala Thr HisTrp Gln Thr Val 225 230 235 240 Ala Gln Thr Thr Asp Glu Arg Val Gln LeuThr Asp Ile Arg Pro Ser 245 250 255 Arg Trp Tyr Gln Phe Arg Val Ala AlaVal Asn Val His Gly Thr Arg 260 265 270 Gly Phe Thr Ala Pro Ser Lys HisPhe Arg Ser Ser Lys Asp Phe Ser 275 280 285 Ala Pro Pro Ala Pro Ala AsnLeu Arg Leu Ala Asn Ser Thr Val Asn 290 295 300 Ser Asp Gly Ser Val ThrVal Thr Ile Val Trp Asp Leu Pro Glu Glu 305 310 315 320 Pro Asp Ile PheVal His His Tyr Lys Val Phe Trp Ser Trp Met Val 325 330 335 Ser Ser LysSer Leu Val Pro Thr Lys Lys Lys Arg Arg Lys Thr Thr 340 345 350 Asp GlyPhe Gln Asn Ser Val Ile Leu Glu Lys Leu Gln Pro Asp Cys 355 360 365 AspTyr Val Val Glu Leu Gln Ala Ile Thr Tyr Trp Gly Gln Thr Arg 370 375 380Leu Lys Ser Ala Lys Val Ser Leu His Phe Thr Ser Thr His Ala Thr 385 390395 400 Asn Asn Lys Glu Gln Leu Val Lys Thr Arg Lys Gly Gly Ile Gln Thr405 410 415 Gln Leu Pro Phe Gln Arg Arg Arg Pro Thr Arg Pro Leu Glu ValGly 420 425 430 Ala Pro Phe Tyr Gln Asp Gly Gln Leu Gln Val Lys Val TyrTrp Lys 435 440 445 Lys Thr Glu Asp Phe Thr Val Asn Arg Tyr His Val ArgTrp Phe Pro 450 455 460 Glu Ala Cys Ala His Asn Arg Thr Thr Gly Ser GluAla Ser Ser Gly 465 470 475 480 Met Thr His Glu Asn Tyr Ile Ile Leu GlnAsp Leu Ser Phe Ser Cys 485 490 495 Lys Tyr Lys Val Thr Val Gln Pro IleArg Pro Lys Ser His Ser Lys 500 505 510 Ala Glu Ala Val Phe Phe Thr ThrPro Pro Cys Ser Ala Leu Lys Gly 515 520 525 Lys Ser His Lys Pro Ile GlyCys Leu Gly Glu Ala Gly His Val Leu 530 535 540 Ser Lys Val Leu Ala LysPro Glu Asn Leu Ser Ala Ser Phe Ile Val 545 550 555 560 Gln Asp Val AsnIle Thr Gly His Phe Ser Trp Lys Met Ala Lys Ala 565 570 575 Asn Leu TyrGln Pro Met Thr Gly Phe Gln Val Thr Trp Ala Glu Val 580 585 590 Thr ThrGlu Ser Arg Gln Asn Ser Leu Pro Asn Ser Ile Ile Ser Gln 595 600 605 SerGln Ile Leu Pro Ser Asp His Tyr Val Leu Thr Val Pro Asn Leu 610 615 620Arg Pro Ser Thr Leu Tyr Arg Leu Glu Val Gln Val Leu Thr Pro Gln 625 630635 640 Gly Glu Gly Pro Ala Thr Ile Lys Thr Phe Arg Thr Pro Glu Leu Pro645 650 655 Pro Ser Ser Ala His Arg Ser His Leu Lys His Arg Ala Pro HisHis 660 665 670 Tyr Lys Pro Ser Pro Glu Arg Tyr 675 680 24 base pairsnucleic acid single linear other nucleic acid /desc = “SYNTHETIC DNA” 2CAGCCAATGG TGCGGCCTCC TGTC 24 22 base pairs nucleic acid single linearother nucleic acid /desc = “SYNTHETIC DNA” 3 TCCCGGCAGA CAGCGACTCC GT 22

What is claimed as new and is desired to be secured by Letters Patent ofthe United States is:
 1. A polypeptide that is an isolated or purifiedfragment of KAL protein of SEQ ID NO: 1 that promotes the survival ofneurons, promotes neurite outgrowth, or induces neurite fasciculation.2. The polypeptide of claim 1 that is glycosylated.
 3. The polypeptideof claim 1 that is not glycosylated.
 4. The polypeptide of claim 1 thatcomprises at least one fibronectin type III repeat.
 5. The polypeptideof claim 1 that comprises residues 182 to 286 of SEQ ID NO:
 1. 6. Thepolypeptide of claim 1 that comprises residues 287 to 403 of SEQ IDNO:
 1. 7. The polypeptide of claim 1 that comprises residues 404 to 541of SEQ ID NO:
 1. 8. The polypeptide of claim 1 that comprises residues542 to 662 of SEQ ID NO:
 1. 9. A composition comprising the polypeptideof claim
 1. 10. A method for promoting the survival of neurons,promoting neurite outgrowth, or inducing neurite fasciculationcomprising administering an amount of the polypeptide of claim 1effective to promote the survival of neurons, promote neurite outgrowth,or induce neurite fasciculation.